PROJECT SUMMARY/ABSTRACT Gross chromosomal rearrangements are a severe class of genomic aberration that has a profound impact on human health. Genomic instability in the form of chromosomal translocations is a well-established driver in many cancers through the activation of oncogenes. Intrachromosomal rearrangements such as interstitial deletions also contribute to cancer by inactivating tumor suppressor genes and to many forms of inherited and de novo germline genetic disease. Because translocations and intrachromosomal rearrangements represent novel recombination junctions between two distant chromosomal regions, each of these mutation types are believed to be the product of inaccurate repair of two DNA double-strand breaks (DSBs). Support for this idea comes from the fact that most chromosomal rearrangements show only microhomology at junctions indicative of an end-to-end rejoining process, as opposed to homologous recombination (HR) which uses long tracts of homology to promote accurate repair. The major ?canonical? nonhomologous end joining (c-NHEJ) pathway of DSB repair dependent on the Ku and DNA ligase IV proteins can suppress rearrangements by promoting repair of DSB ends in cis, i.e. by joining the two ends resulting from a single DSB, but is also often implicated in rearrangement through the trans joining of distant ends. Other less well-described ?alternative? NHEJ (a- NHEJ) pathways also exist which are even more error prone. A critical feature that influences the balance of these various repair pathways (c-NHEJ, a-NHEJ, and HR) and outcomes (accurate, local mutations, and rearrangements) at DSBs is the processing of DNA ends via nucleolysis and polymerization. Limited processing makes dirty or incompatible DSB ends ready for ligation in c-NHEJ. More extensive processing exposes microhomologous annealing surfaces for a-NHEJ. Still more extensive processing allows Rad51 assembly and strand invasion in HR. However, there are many fundamental questions about end processing that limit our ability to understand this critical regulated disposition of DSBs. Moreover, given that both cis and trans repair of DSBs require seemingly similar processing for junctions to be formed, there must be further as yet poorly understood properties of DSBs and their chromosomal locations that influence this disposition. The goals of this project are to explore with base-pair precision the processing of DSB ends during normal and mutagenic NHEJ (Aim 1) and to determine the genomic factors that shape the contribution of these processes to chromosomal rearrangements (Aim 2). Because of the need to achieve tight control over the formation, kinetics, and locations of DSBs, this work will be executed in the highly manipulable yeast genetic system. It will further be made possible by innovative applications of new and enabling high-throughput sequencing technologies. Upon completion of these studies, we will have a better understanding of the molecular mechanisms that both suppress and promote chromosome rearrangement through NHEJ. 1